Galphaq protein variants and their use in the analysis and discovery of agonists and antagonists of chemosensory receptors

ABSTRACT

The invention provides a series of Gα q  protein variants that functionally couple to sensory cell receptors such as taste GPCRs (TRs) and olfactory GPCRs (ORs) in an overly promiscuous manner. According to the invention, the functional coupling can be determined, for example, by measuring changes in intracellular IP3, or calcium. In a particular embodiment, the Gα q  protein variants can be expressed in mammalian cell lines or  Xenopus  oocytes, and then evaluated using calcium fluorescence imaging and electrophysiological recording.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/984,292 filed Oct. 29, 2001, which claims priority to U.S.Provisional Application No. 60/243,770, filed on Oct. 30, 2000, and areincorporated herein by reference in their entirety.

FIELD OF INVENTION

This invention relates to Gαq protein variants and their use in theanalysis and discovery of agonists and antagonsists of chemosensoryreceptors, such as G protein coupled receptors involved in sensing oftastants, olfactants, and pheromones.

BACKGROUND OF INVENTION

Heterotrimeric G proteins, consisting of alpha, beta and gamma subunits,couple ligand-bound seven transmembrane domain receptors (GPCRs orG-protein coupled receptors) to the regulation of effector proteins andproduction of intracellular second messengers such as cAMP, cGMP, andCa²⁺. G protein signaling mediates the perception of environmental cuesin all higher eukaryotic organisms, including yeast, Dictyostelium,plants, and animals. Agonist-bound sensory receptors catalyze theexchange of GTP for GDP on the surface of the Gα subunit to initiateintracellular responses to extracellular signals. Intracellularsignaling is mediated through various effector enzymes, including cGMPphosphodiesterase, phospholipase C, adenylate cyclase, etc. (seeKinnamon & Margolskee, 1996, Curr. Opinion Neurobiol. 6: 506-513). Mosteffector proteins interact with the Gα, although Gβγ subunits alsocontribute to the specificity of receptor-G protein coupling (Xu et al.,1998, J. Biol. Chem. 273(42): 27275-79).

The G protein α subunits are grouped into four families, Gα_(s), Gα_(i),Gα_(q), and Gα₁₂ according to their sequence homologies and functionalsimilarities. The Gα_(q) family members couple a large group of GPCRs tophospholipase C. Activation of Gα_(q) coupled GPCRs inducesintracellular calcium release and the capacitative calcium entry fromextracellular space. The consequential increase of cytosolic calciumconcentration can be effectively detected by using synthetic orgenetically-engineered fluorescent calcium indicators, bioluminescentcalcium indicators, calcium-activated ion currents, and by monitoringcalcium-regulated gene transcription. Assays based on such calciumreadout are available in high-throughput screening (HTS) format.

Signaling specificity among α subunits of the same class having similarbiochemical functions is not well understood in vivo. For instance, theGα_(q) (G_(q)) class includes four proteins expressed in mammals, calledGα_(q), Gα₁₁, Gα₁₄, and Gα₁₅ (in mice, Gα₁₆ in humans). Whereasorthologs of these subunits are highly conserved across species (99, 97,96 and 85% identity, respectively), paralogs of these subunits(expressed in the same species) are not as conserved. This suggests thateach type of subunit in the G_(q) class has a distinct function,however, when transfected into Sf9 cells, the subunits stimulatedphospholipase C with similar potency and showed similar activities(Nakamura et al., 1995, J. Biol. Chem. 270: 6246-6253). Xu andcolleagues subsequently showed by gene knockouts in mice that Gq_(α)subunits promiscuously couple to several different receptors in variouscell types (1998, J. Biol. Chem. 273(42): 27275).

The promiscuity of the Gα_(q) subclass of G protein subunits provides avaluable tool for analyzing the role of G protein complexes and GPCRs inchemosensory transduction. For instance, the ability of Gα_(q) proteinsto bypass the selectivity of the receptor G-protein interaction can beused to study the molecular mechanism of receptor-induced G-proteinactivation. In addition, the promiscuity toward receptors may be helpfulin identifying ligands corresponding to orphan receptors whose signalingproperties are unknown. Promiscuous G protein subunits play aparticularly useful role in generating screening assays for highaffinity GPCR agonists, antagonists, and modulators of chemosensoryactivity, in that using a single G protein coupler removes thevariability of the G protein from the equation, thereby simplifyinginterpretation of results gleaned from various modulating compounds andGPCRs. Chemosensory modulating compounds involved in taste and/or smell,for instance, could then be used in the pharmaceutical and foodindustries to customize taste or aroma. In addition, such chemosensorymolecules could be used to generate topographic maps that elucidate therelationship between the taste cells of the tongue or olfactoryreceptors (Ors) and sensory neurons leading to the brain.

Despite their promiscuity, however, Gα_(q) class subunits do not mediateall GPCR—effector interactions. For instance, human Gα₁₆ and its murinecounterpart Gα₁₅ are promiscuous G proteins in that they couple to GPCRsof different G protein families (Offermanns and Simon, 1995; Negulescuet al., 1997). However, they are not true universal adapters for GPCRsin that there are at least 11 GPCRs reported to be incapable ofactivating Gα₁₅/Gα₁₆ (Wu et al., 1992; Arai et al., 1996; Kuang et al.,1996; Lee et al., 1998; Parmentier et al., 1998; Mody et al., 2000).Similar problems arise when using Gα₁₅/α₁₆ to identify ligands of ORsand T2Rs (bitter taste receptors) in that (1) calcium responses toodorants are small and quickly desensitized for ORs in Gα₁₅/α₁₆transiently transfected cells (Krautwurst et al., 1998); (2) most T2Rsremain orphan using cell lines stably transfected with Gα₁₅ (Adler etal., 2000; Chandrashekar et al., 2000); and (3) threshold concentrationof denatonium measured is at least one order higher than expected forbitter receptors, hT2R4 and mT2R8 expressed in cells stably transfectedwith Gα₁₅ (Adler et al., 2000; Chandrashekar et al., 2000). Theseproblems suggest that the coupling efficiency between ORs/T2Rs andGα₁₅/α₁₆ is weak and may vary within the family of ORs and T2Rs.

Given the partial promiscuity of Gα_(q) class proteins, it would bedesirable to identify or create Gα protein subunits that are morepromiscuous than their native counterparts, and which are capable ofinteracting with a wider variety GPCRs.

SUMMARY OF INVENTION

The present invention addresses the above described problems associatedwith using Gα₁₅/α₁₆, as well as other problems known in the art relatingto the use of weakly promiscuous Gα proteins. Generally, the inventionprovides a series of Gα_(q) (G_(q) class) protein variants thatfunctionally couple sensory cell receptors such as taste GPCRs (TRs) andolfactory GPCRs (ORs). According to the invention, the functionalcoupling can be determined, for example, by measuring changes inintracellular IP3 or calcium. In a particular embodiment, the G_(q)protein variants can be expressed in mammalian cell lines or Xenopusoocytes, and then evaluated using calcium fluorescence imaging andelectrophysiological recording.

In one aspect of the invention, G alpha class q (G_(q)) variants thatare capable of widely promiscuous functional coupling to chemosensoryreceptors, such as taste and olfactory receptors, and isolated nucleicacid sequences encoding the same are provided. Another aspect of theinvention is directed to chimeric G_(q) variants and the isolatednucleic acids encoding the same. In one embodiment, the chimeric G_(q)protein variants comprise C-terminal sequences from transducin orGα_(olf), which exhibit improved functional coupling to taste andolfactory receptors, respectively.

In yet another aspect of the invention, a method for the analysis anddiscovery of agonists and/or antagonists of chemosensory receptors usingthe G_(q) protein variants is provided. One embodiment is directed to amammalian cell-based assay using a transiently transfected gene or cDNAencoding a G_(q) protein variant. Another embodiment is directed to amammalian cell-based assay using a stably expressed gene or cDNAsencoding G_(q) protein variants. In yet another embodiment, a method foranalysis and discovery of agonists and/or antagonists of chemosensoryreceptors in Xenopus oocytes using genes, RNAs or DNAs encoding G_(q)protein variants is provided. The agonists and/or antagonists discoveredusing the disclosed assays are also encompassed, as are antibodies whichbind specifically to the G_(q) variants described herein, but not thosewhich also bind to known G_(q) proteins.

Other aspects of the invention relate to expression vectors comprisingnucleic acid sequences encoding the G_(q) protein variants of theinvention, as well as host cells comprising such expression vectors.Further aspects of the invention will become apparent to one of skill inthe art from the following detailed description and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the alignment of amino acid sequences of humanGα_(q), Gα_(s) and Gα₁₆ by the Clustal method.

FIG. 2 illustrates the amino acid sequences of mouse and human Gα_(q).Significant amino acids described herein are boxed and differencesbetween human and mouse are underlined.

FIG. 3 illustrates the amino acid sequences of mouse and human Gα_(q)proteins according to the invention. The variations of the amino acidsof Gα_(q) are depicted in parenthesis. The sequence numbers of aminoacid H or Q, V or L are 28 and 29 respectively. The sequence number ofamino acid G or D is 66. Truncation of N-terminal six amino acids(MTLESI) are shown as ΔN. Hemaglutinin (HA) epitope tag (DVPDYA) spansfrom 125 to 130. C-terminal five amino acids (-t5) or 44 amino acids(-t44) of transducin and five amino acids of Gα_(olf) (-olf5) are usedrespectively to replace those of Gα_(q).

FIG. 4 illustrates additional amino acid sequences according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

As described above, there are known problems with the use of Gα₁₅/Gα₁₆to couple chemosensory receptors in that the coupling efficiency betweenORs/T2Rs and Gα₁₅/α₁₆ is weak and may vary within the family of ORs andT2Rs (bitter taste receptors). As such, the invention provides a seriesof G_(q) protein variants that functionally couple sensory cellreceptors such as taste GPCRs (TRs) and olfactory GPCRs (ORs) in apromiscuous manner. According to the invention, “promiscuous” or“promiscuity” refers to the ability to functionally couple to more thanone taste GCPRs and/or olfactory GPCRs. “Increased promiscuity” or“widely” promiscuous refers to the ability to functionally couple tomore taste GCPRs and/or olfactory GPCRs than would be demonstrated bythe native G_(q) protein.

The term “G_(q)” as used herein encompasses all the Gα_(q) subclasses,including Gα_(q), Gα₁₁, Gα₁₄, and Gα₁₅ (in mice, Gα₁₆ in humans).However, the chimeric promiscuous or widely promiscuous G_(q) proteinsdescribed herein may have sequences incorporated from other Gα classproteins, for instance, from Gα_(s), Gα_(i) and Gα₁₂. The existingvariation between members of the G_(q) class could be utilized incombination with the characteristic of promiscuity to generatepromiscuous G_(q) proteins having altered or new receptor specificities.Protein sequence similarity between Gα and Gα15/Gα16 is less than 57%(FIG. 1). Accordingly, such high divergence should result in significantdifferences in efficiency and selectivity of receptor coupling. Theidentification of functionally active Gq protein variants could allowfor the pharmacological and genetic modulation of sensory transductionpathways.

For example, Gq protein variants could enable screening for highaffinity agonists, antagonists, inverse agonists, and other modulatorsof sensory cell transduction and activity. Such sensory cell modulatorscould then be used in the pharmaceutical and food industries tocustomize sensory perceptions. In addition Gq protein variants couldserve as tools in the generation of sensory topographic maps.

According to the invention, Gq protein variants include variants havingpoint mutations that increase promiscuity with regard to GPCR coupling.For instance, the inventors have found that mouse Gα_(q) variantscomprising a Gly to Asp change at position 66 (G66D) demonstrateincreased promiscuity. Similar mutations are predicted to have a similareffect on the activity of the corresponding human Gα₁₆ subunit given thelevel of homology and similar activity demonstrated between the twoproteins. The mutation G66D is localized at linker 1 region betweenhelices α1 and αA of Gα_(q) (Lambright et al., 1996). For reference, theamino acid sequences for mouse and human Gα_(q) are listed in FIG. 2.

It was found by functional analysis using single-cell calcium imagingthat activation of multi-family GPCRs evoked increases in cytosoliccalcium in the presence of the Gα_(q) variants with the G66D mutation.These GPCRs include Gα_(s)-coupled β-adrenergic receptor,Gα_(olf)-coupled mouse 17 olfactory receptor, Gα_(i)-coupled m2muscarinic receptor, and gustducin-coupled bitter receptor mT2R5. Nosignificant change in cytosolic calcium could be detected by activationof the above GPCRs in the absence of the Gα_(q) variants. AdditionalGPCRs can include those disclosed in U.S. patent application Ser. No.09/510,332 filed Feb. 22, 2000; and U.S. Provisional Application Nos.60/213,849 filed Jun. 23, 2000; 60/209,840 filed Jun. 6, 2000;60/195,536 filed Apr. 7, 2000; 60/195,534 filed Apr. 7, 2000; 60/195,532filed Apr. 7, 2000; which are herein incorporated by references for allpurposes in a manner consistent with this disclosure.

Thus, Gα_(q) variants according to the invention can comprise amino acidsubstitutions at or near position 66, or at any other position thatresults in an increase of promiscuity by the variant Gα protein. OtherG_(q) subclass variants can be designed having similar mutations.Mutations can be identified and isolated using site directed or randommutagenesis according to techniques that are known in the art, includingrandom saturation mutagenesis around the mutation sites describedherein. The variants may comprise these one or more of these mutationsalone or in combination with C-terminal substitutions. In anotherembodiment of the invention, Gα_(q) and other G_(q) subclass variantscomprise C-terminal sequences derived from other G proteins.

For instance, the present inventors have also discovered that the Gly toAsp mutation is synergistic with the replacement of the C-terminus ofGα_(q) by that of transducin or Gα_(olf). Gα_(q) proteins containingC-terminal amino acids from transducin or Gα_(olf) in combination with aGly66 to Asp alteration show increased activity compared to individualchimeras alone. A preferred embodiment is a variant G_(q) proteinshaving at least about five amino acids in the C terminus of said G_(q)protein replaced by at least about five amino acids from the C terminusof Gα_(olf) or transducin, wherein said C-terminal substitutionincreases promiscuity of said variant G_(q) protein as compared to thecorresponding native G_(q) protein. Up to 44 amino acids of the Cterminus of transducin or Gα_(olf) may be incorporated. Other possiblevariants are shown in FIGS. 3 and 4.

Other mutations and substitutions are envisioned to be within the scopeof the invention. For instance, it would be within the level of skill inthe art to perform additional amino acid substitutions at other aminoacid positions using known protocols of recombinant gene technologyincluding PCR, gene cloning, site-directed mutagenesis of cDNA,transfection of host cells, and in-vitro transcription. The variantscould then be screened for functional coupling to chemosensory receptorsas described herein. Further, additional C-terminal substitutions couldbe made from other G-protein molecules known in the art.

The present invention also includes isolated Gα_(q) subunit polypeptidevariants comprising polypeptides with greater than 80% amino acidsequence identity to a sequence selected from the group consisting ofSEQ ID Nos 1-26. More preferably, variants comprising polypeptides withgreater than 90% amino acid sequence identity are included, with themost preferable homologs being at least about 95% identical to thevariants described herein.

The invention also includes isolated nucleic acid sequences encoding theG_(q) protein variant polypeptides of the invention. Included areisolated nucleic acid sequences comprising a nucleic acid encoding apolypeptide with greater than 80% amino acid sequence identity to asequence selected from the group consisting of SEQ ID Nos 1-26. Morepreferably, the isolated nucleic acid sequence encoding a Gα_(q) proteinvariant comprises a nucleic acid encoding a polypeptide with greaterthan 90% amino acid sequence identity to a sequence selected from thegroup consisting of SEQ ID Nos 1-26. Most preferred are isolated nucleicacid sequences encoding Gα_(q) protein variants which comprise a nucleicacid encoding a polypeptide with greater than about 95% amino acidsequence identity to a sequence selected from the group consisting ofSEQ ID Nos 1-26.

The terms “identical” or “percent identity” in the context of two ormore protein or nucleic acid sequences refers to sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same when compared and alignedfor maximum correspondence over a comparison window or designatedregion, using either a sequence comparison algorithm that is known inthe art or by manual inspection. Sequences with over 80% sequenceidentity are said to be “substantially identical.” Optionally, theidentity, exists over a region that is at least about 25-30 amino acidsor nucleotides in length, or optionally over a region that is 75-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. A “comparisonwindow” as used herein includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 500, usually about 50 to about 200, more usually about 100 to150 in which a sequence may be compared to a reference sequence of thesame number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences are well known inthe art (see, e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2: 482,Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85: 2444, and Current Protocols inMolecular Biology, Ausubel et al., 1995 Suppl.).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity,and can be obtained from the GCG sequence analysis software package,e.g., version 7.0 (Devereaux et al., 1984, Nuc. Acids Res. 12: 387-395).Another example of an algorithm that is suitable for determining percentsequence identity is the BLAST or BLAST 2.0 algorithm described inAltschul et al., 1977, Nuc. Acids Res. 25: 3389-3402 (1977) and Altschulet al., 1990, J. Mol. Biol. 215: 403-410, respectively. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Also included in the present invention are antibodies that selectivelybind to the variant G_(q) alpha proteins described herein, but not tothe corresponding native G_(q) alpha protein. Such antibodies includewhole, chimeric, humanized, tetramer, single chain, domain-deleted andother recombinant antibodies of any immunglobulin class, as well asantibody fragments, Fv, Fab′, (Fab)′₂, etc. Preparation of suchantibodies may be performed using any method known in the art (see,e.g., Kohler and Milstein, 1975, Nature 256: 495-97; Kozbar et al.,1983, Immunology Today 4: 72; Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, 1985). Mice or other animals may beimmunized with the G_(q) protein variants of the invention in order togenerate antibodies, which may be screened to identify those specificfor the G_(q) variants of the invention which also do not recognize thecorresponding native G_(q) protein.

The present invention also encompasses expression vectors comprising thenucleic acid sequences of the present invention operably linked to apromoter that functions in mammalian cells or Xenopus oocytes. A“promoter” is defined as an array of nucleic acid control sequences thatdirect transcription of a nucleic acid. A “promoter” includes all thenecessary sequences near the start site of transcription, i.e.,including a polymerase binding site. A promoter optionally includesdistal enhancer or repressor elements which can be located as much asthousands base pairs away from the start site of transcription.Promoters may be either constitutive, i.e. active under mostenvironmental and developmental conditions, or inducible, i.e., underspecific environmental or developmental control. The term “operablylinked” refers to a functional linkage between a nucleic acid expressioncontrol sequence (such as a promoter) and a second nucleic acidsequence, such as one encoding a variant G_(q) protein as described inthe present invention, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.An “expression vector” is a nucleic acid construct comprising a codingnucleic acid sequence according to the invention operably linked to apromoter, which allows for recombinant production of the variant Gqproteins described herein. Expression vectors encompassed by theinvention can be either incorporated into the genome of a host cellafter transfection, or replicate extrachromosomally. Expression vectorscan be either plasmids, viruses or nucleic acid fragments.Alternatively, coding sequences can be incorporated into the genomebehind a native promoter, thereby creating an operable expressionlinkage following transfection. Host cells transfected with theexpression vectors of the invention are also encompassed.

The present invention also includes methods for identifying a compoundthat modulates sensory signaling in sensory cells, the method comprisingthe steps of: (1) contacting the compound with a cell expressing theG_(q) variant protein according to claim 1; and (2) determining thefunctional effect of said compound upon the G_(q) protein variant.Typically, a cell expressing said G_(q) variant protein is a transfectedsensory cell, or other transfected cell suitable for making functionalmeasurements of G protein activity, i.e., Xenopus oocyte. Functionaleffects of possible modulatory compounds may be determined by measuringchanges in intracellular IP3 or Ca²⁺. Functional effects may also bedetermined by measuring changes in the electrical activity of the cellsexpressing said G_(q) variant protein or by observing modification of anintracellular effector enzyme. Possible modulatory compounds includeagonists, antagonists, antibodies, small molecules and proteins.

Also included in the invention are methods for identifying a compoundthat interacts with the G_(q) variant protein of claim 1, comprising thesteps of (1) contacting said G_(q) variant protein with a test compound;and (2) detecting a binding interaction between said compound and saidG_(q) protein variant. Methods of detecting the binding of G_(q) proteinvariants to compounds can be performed wherein said G_(q) variantprotein is linked to solid phase, either covalently or noncovalently.

The present invention also includes an artificial array of GPCRsfunctionally coupled to the G_(q) variant of claim 1, wherein said arrayis a model of a native arrangement of GPCRs. For instance, the nativearrangement can be an arrangement of olfactory receptors (ORs) typicallyseen in a mammalian nose, or an arrangement of taste receptors typicallyseen on a mammalian tongue. Said taste receptors typically include atleast one type of taste receptor selected from the group consisting ofbitter, sweet, salty, unami and sour taste receptors, in light of theobservations that such taste receptors are typically arranged inspatially organized manner. The artificial arrays of the presentinvention are useful for analyzing the response to different sensorycompounds in relation to brain activity. Such arrays will be improved bythe promiscuous variant G_(q) proteins of the present invention, whichwill simplify interpretation of results that might normally becomplicated by the requirement for different G protein subunits forevery GPCR in such an array.

It is also envisioned that the G_(q) protein variants of the inventioncould be used in other types of functional assays such as biochemicalbinding assays, enzymatic assays, other cell-based assay, as well aswith in vivo systems such as transgenic mice.

The following examples serve merely to illustrate the invention, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES

Initially, the Gα_(q) variant protein termed mG_(q) (ON-HVD-HA) of SeqID#5, described in Kostenis et al. (1998), was shown to functionallycouple to taste receptor mT2R5 and olfactory OR 17. Previously, it hadnot been known whether this protein would allow functional expression ofchemosensory receptors such as taste and olfactory receptors. Theresponse using the Kostenis et al G protein was weak. Therefore, inorder to improve functional coupling a series of variants was created.The variants were chimeras between the variants of the Kostenis Gprotein which contained the C terminal sequences from Gα_(olf) ortransducin. Variants containing the C-terminal changes exhibitedimproved function. The use of C-terminal replacements in G proteins hadpreviously been reported by Conklin (Conklin et al., 1993; Conklin etal., 1996; Coward et al., 1999) but the sequences from Gα_(olf) ortransducin had not previously been shown to function with taste orolfactory receptors.

A series of Gα_(q) protein variants having the sequences listed in FIG.3 were constructed and tested in mammalian cell-based systems and inXenopus oocytes for functional coupling efficiency with bitter receptormT2R5 and mouse olfactory receptor 17. As shown in Table I below, oneset of G proteins function with a taste receptor and another set of Gproteins function with an olfactory receptor. All active G proteinsconsisted of mouse sequences, however given the similarities betweenhuman and mouse Gα_(q) proteins, it is anticipated that the humanvariants will also functionally couple with human chemosensoryreceptors. TABLE I Functional Activity of Gq Variants FunctionalFunctional Activity With Activity Taste Receptor with Olfactory GqVariants Seq ID # MT2R5 Receptor ml7 MGq 1 − N/A MGq(ΔN) 2 − N/A MGq(HA)3 − N/A MGq(ΔN-HA) 4 − N/A MGq(ΔN-HVD-HA) 5 + + MGq(ΔN-HVD-HA)-t5 6 ++N/A MGq(ΔN-HVD-HA)-t44 7 ++ N/A MGq(ΔN-HV-HA) 8 − N/A MGq(HV-HA) 9 − N/AMGq(D-HA) 10 + N/A MGq(HVD-HA) 11 + N/A MGq(HV-HA)-t5 12 + N/AMGq(HVD-HA)-t5 13 ++ N/A MGq(ΔN-HVD-HA)-olf5 14 N/A ++ HGq 15 − N/AHGq(ΔN) 16 − N/A+ means functionally couples with chemosensory receptor++ means functionally couples with chemosensory receptor− means does not functionally coupleN/A mean not testedMaterials and Methods

The Ga15 chimeras were generated by PCR with mutagenic 3′ primers. Thesequence of our Ga15 clone corresponds to databank sequences (e.g.,accession BC005439) except for a silent single nucleotide polymorphismshown in bold underline below. The last six codons of Ga15 and thesequences they were replaced with are shown in italic underline below.The Ga15 chimeras were generated with 5′ AscI sites (GGCGCGCCgcc joinedto the start ATG) and 3′ NotI sites (GCGGCCGC joined to the stop TGA)and cloned as AscI-NotI fragments in the AscI-NotI polylinker sites ofthe pEAK10 expression vector (Edge Biosystems). Ga 15 (SEQ ID NO:27)Atggcccggtccctgacttggggctgctgtccctggtgcctgacagaggaggagaagactgccgccagaatcgaccaggagatcaacaggattttgttggaacagaaaaaacaagagcgcgaggaattgaaactcctgctgttggggcctggtgagagcgggaagagtacgttcatcaagcagatgcgcatcattcacggtgtgggctactcggaggaggaccgcagagccttccggctgctcatctaccagaacatcttcgtctccatgcaggccatgatagatgcgatggaccggctgcagatccccttcagcaggcctgacagcaagcagcacgccagcctagtgatgacccaggacccctataaagtgagcacattcgagaagccatatgcagtggccatgcagtacctgtggcgggacgcgggcatccgtgcatgctacgagcgaaggcgtgaattccaccttctggactccgcggtgtattacctgtcacacctggagcgcatatcagaggacagctacatccccactgcgcaagacgtgctgcgcagtcgcatgcccaccacaggcatcaatgagtactgcttctccgtgaagaaaaccaaactgcgcatcgtggatgttggtggccagaggtcagagcgtaggaaatggattcactgttttgagaacgtgattgccctcatctacctggcctccctgagcgagtatgaccagtgcctagaggagaacgatcaggagaaccgcatggaggagagtctcgctctgttcagcacgatcctagagctgccctggttcaagagcacctcggtcatcctcttcctcaacaagacggacatcctggaagataagattcacacctcccacctggccacatacttccccagcttccagggaccccggcgagacgcagaggccgccaagagcttcatcttggacatgtatgcgcgcgtgtacgcgagctgcgcagagccccaggacggtggcaggaaaggctcccgcgcgcgccgcttcttcgcacacttcacctgtgccacggacacgcaaagcgtccgcagcgtgttcaaggacgtgcgggactcggtgctggcccggtacctggacgagatcaacctgctgtga GACTGTGGCCTCTTCTGA Gai1 (SEQ ID NO:28)GAGTACAATCTGGTCTGA Gaq (SEQ ID NO:29) CAGTATGAGCTCTTGTGA Gas (SEQ IDNO:30) GAGTGCGGCCTCTACTGA Gai3 (SEQ ID NO:31) GGATGCGGACTCTACTGA Gao(SEQ ID NO:32) TACATCGGCCTCTGCTGA Gaz (SEQ ID NO:33) GACATCATGCTCCAATGAGa12 (SEQ ID NO:34) CAACTAATGCTCCAATGA Ga13 (SEQ ID NO:35)CACCAGGTTGAACTCTGA Ga14 (SEQ ID NO:36)

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1-39. (canceled)
 40. An isolated nucleic acid sequence encoding a G_(q)protein variant wherein at least the last five amino acids are replacedby the 5 C-terminal amino acids of Gα_(olf) or transducin and theresultant altered G_(q) variant exhibits greater promiscuity compared tothe corresponding native G_(q) protein.
 41. An isolated nucleic acidsequence encoding the G_(q) protein variant of claim 40, wherein thenative G_(q) protein is a human G_(q) protein.
 42. An isolated nucleicacid sequence encoding the G_(q) protein variant of claim 40, whereinthe native G_(q) protein is a mouse G_(q) protein.
 43. An isolatednucleic acid sequence encoding a G_(q) protein variant comprising anucleic acid encoding a polypeptide with at least 95% amino acidsequence identity to a sequence selected from the group consisting ofSEQ ID NOS 1-26.
 44. An isolated nucleic acid sequence encoding a Gα_(q)protein variant comprising a nucleic acid encoding polypeptide withgreater than 95% amino acid sequence identity to a sequence selectedfrom the group consisting of SEQ ID Nos 1-26.
 45. An isolated nucleicacid sequence encoding a Gα_(q) protein variant that is encased by aamino sequence selected from the group consisting of SEQ ID Nos 1-26.46. An expression vector comprising the nucleic acid sequence of claim40 operably linked to a promoter that functions in mammalian cells orXenopus oocytes.
 47. An expression vector comprising the nucleic acidsequence of claim 41 operably linked to a promoter that functions inmammalian cells or Xenopus oocytes.
 48. An expression vector comprisingthe nucleic acid sequence of claim 42 operably linked to a promoter thatfunctions in mammalian cells or Xenopus oocytes.
 49. An expressionvector comprising the nucleic acid sequence of claim 43 operably linkedto a promoter that functions in mammalian cells or Xenopus oocytes. 50.An expression vector comprising the nucleic acid sequence of claim 44operably linked to a promoter that functions in mammalian cells orXenopus oocytes.
 51. An expression vector comprising the nucleic acidsequence of claim 45 operably linked to a promoter that functions inmammalian cells or Xenopus oocytes.
 52. A host cell comprising theexpression vector of claim
 46. 53. A host cell comprising the expressionvector of claim
 47. 54. A host cell comprising the expression vector ofclaim
 48. 55. A host cell comprising the expression vector of claim 49.56. A host cell comprising the expression vector of claim
 50. 57. A hostcell comprising the expression vector of claim 51.